The present invention relates to methods for fabricating solid supports. More specifically, the present invention features methods for fabricating solid supports for in situ synthesis and for carrying out large numbers of reactions. The present invention also features solid supports with in situ synthesized long polynucleotides.
Intense efforts are under way to map and sequence the human genome and the genomes of many other species. In June 2000, the Human Genome Project and biotech company Celera Genomics announced that a rough draft of the human genome has been completed. This information, however, will only represent a reference sequence of the human genome. The remaining task lies in the determination of sequence variations, such as mutations and polymorphism, which is important for the study, diagnosis, and treatment of genetic diseases.
In addition to the human genome, the mouse genome is also being sequenced. Genbank provides about 1.2% of the 3-billion-base mouse genome and a rough draft of the mouse genome is expected to be available by 2003 and a finished genome by 2005. The Drosophila Genome Project has also been completed recently.
During the past decade, the development of array-based hybridization technology has received great attention. This high throughput method, in which hundreds to thousands of polynucleotide probes immobilized on a solid surface are hybridized to target nucleic acids to gain sequence and function information, has brought economical incentives to many applications. See, e.g., McKenzie, S., et al., European Journal of Human Genetics 416-429 (1998), Green et al., Curr.Opin. in Chem. Biol. 2:404-410 (1998), Gerhold et al., TIBS, 24:168-173 (1999), and U.S. Pat. Nos. 5,700,637, 6,054,270, 5,837,832, 5,744,305, and 5,445,943.
One application is the monitoring of gene expression level and comparing of gene expression patterns. Many gene-specific polynucleotide probes derived from the 3xe2x80x2 end of RNA transcripts are spotted on a solid surface. This array is then probed with fluorescently labeled cDNA representations of RNA pools from test and reference cells. The relative amount of transcript present in the pool is determined by the fluorescent signals generated and the level of gene expression is compared between the test and the reference cell. See, e.g., Duggan, D., et al., Nature Genetics Supplement 21:10-14 (1999), DeRisi, J., et al., Science 278:680-686 (1997), and U.S. Pat. Nos. 5,800,992, 5,871,928, and 6,040,138.
Another application of the array technology is the genotyping of mutations and polymorphisms, also known as re-sequencing. Typically, sets of polynucleotide probes, that differ by having A, T, C, or G substituted at or near the central position, are fabricated and immobilized on a solid support by in situ synthesis. Fluorescently labeled target nucleic acids containing the expected sequences will hybridize best to perfectly matched polynucleotide probes, whereas sequence variations will alter the hybridization pattern, thereby allowing the determination of mutations and polymorphic sites. See, e.g., Wang, D., et al., Science 280:1077-1082 (1998) and Lipshutz, R., et al., Nature Genetics Supplement 21:20-24 (1999), and U.S. Pat. Nos. 5,858,659, 5,856,104, 5,968,740, and 5,925,525.
Another application of the array technology is the de novo sequencing of target nucleic acids by polynucleotide hybridization. For example, an array of all possible 8-mer polynucleotide probes may be hybridized with fluorescently labeled target nucleic acids, generating large amounts of overlapping hybridization data. The reassembling of this data by computer algorithm can determine the sequence of target nucleic acids. See, e.g., Drmanac, S. et al., Nature Biotechnology 116:54-58 (1998), Drmanac, S. et al. Genomics 4:114-28 (1989), and U.S. Pat. Nos. 5,202,231, 5,492,806, 5,525,464, 5,667,972, 5,695,940, 5,972,619, 6,018,041, and 6,025,136.
In many applications of the array technology, polynucleotide probes are prepared by in situ synthesis on a solid support in a step-wise fashion. With each round of synthesis, nucleotide building blocks are added to growing chains until the desired sequence and length are achieved at each in situ synthesis site. In general, in situ polynucleotide synthesis on a solid support may be achieved by two general approaches.
First, photolithography may be used to synthesize polynucleotides on a solid support. Ultraviolet light may be shone through holes of a photolithograhic mask onto the support surface, which removes a photoactive protecting group, resulting in a 5xe2x80x2 hydroxy group capable of reacting with another nucleoside. The mask therefore predetermines which nucleotides are activated. Successive rounds of deprotection and chemistry result in polynucleotides with increasing length. This method is disclosed in U.S. Pat. Nos. 5,143,854, 5,412,087, 5,445,934, 5,489,678, 5,571,639, 5,744,305, 5,837,832, 5,871,928, 5,889,165, and 6,040,138.
The second approach is the xe2x80x9cdrop-on-demandxe2x80x9d method, which uses technology analogous to that employed in ink-jet printers (U.S. Pat. Nos. 5,985,551, 5,474,796, 5,700,637, 6,054,270, 6,028,189, 5,927,547, WO 98/41531, Blanchard et al., Biosensors and Bioelectronics 11:687-690 (1996), Schena et al., TIBTECH 16:301 -306 (1998), Green et al., Curr. Opin. Chem. Biol. 2:404-410 (1998), and Singh-Gasson, et al., Nat. Biotech. 17:974-978 (1999)). This approach typically utilizes piezoelectric or other forms of propulsion to transfer reagents from miniature nozzles to solid surfaces. For example, a printer head may travel across the array, and at each spot, electric field contracts, forcing a microdroplet of reagents onto the array surface. Following washing and deprotection, the next cycle of polynucleotide synthesis is carried out.
The photolithography method used for in situ synthesis of polynucleotide probes presents many problems. First, the highest step yield reported for in situ photolithographic synthesis of polynucleotides is about 90% (Forman, J., et al., Molecular Modeling of Nucleic Acids, Chapter 13, p. 221, American Chemical Society (1998) and McGall et al., J. Am. Chem. Soc. 119:5081-5090 (1997)). In other words, when an additional nucleotide is added to each in situ synthesis site on a solid support, only about 90% of the products contain sequences with the additional nucleotide. The remaining 10% of the products are no longer available for nucleotide extension and remain as truncated sequences. The step yield of 90% is rather low for polynucleotide synthesis and is especially problematic for long polynucleotide synthesis. If one were to make polynucleotides of 50 nucleotides long with an average step yield of 90%, essentially none (only about 0.6%) on each in situ synthesis site would have the desired length of all 50 nucleotides. Essentially all polynucleotides (about 99.4%) would be truncated to some lesser number of nucleotides in length. If one were to make polynucleotides of 30 nucleotides long with an average step yield of 90%, less than about 5% of polynucleotides on each in situ synthesis site would have the desired length of 30 nucleotides and the rest (about 95%) being truncated sequences. Because truncated sequences do not correspond to the desired full length probe sequence, they may bind to unintended target nucleic acids and generate false hybridization signals. Therefore, the presence of large amounts of truncated polynucleotides inevitably leads to unpredictable hybridization performance.
Second, photolithographic synthesis of polynucleotides is expensive (e.g., the cost of masks). An array with N-mer polynucleotide probes may require 4xc3x97N photolithographic masks. For example, photolithographic synthesis of a 25-mer typically requires repeating the deprotection and coupling cycle about 100 times with about 100 different masks. To lower the cost per array, one has to synthesize large numbers of arrays with the same masks and probe sequences. In addition, changing the polynucleotide probe length and base composition in photolithography requires changing masks, which leads to high cost.
Finally, it is often necessary to introduce unnatural polynucleotide probes, for example, to balance the stability difference between A/T rich and G/C rich sequences, or to introduce a cleavage site. Incorporation of unnatural structures into the polynucleotide probes in the photolithography method involves new photodeprotection chemistry and will likely encounter even lower step yields and high redesigning cost. In addition, the photolithographic in situ synthesis method currently allows polynucleotide synthesis only in the 3xe2x80x2 to 5xe2x80x2 direction.
There is a need in the art to develop methods for fabricating solid supports for use in in situ synthesis of long polynucleotide probes with high efficiency and low cost. The present invention features the in situ synthesis of polynucleotides longer than 30 nucleotides. The present invention also features the in situ synthesis of polynucleotides longer than 15 nucleotides with greater purity. The average step yield is near or above 98%. The present synthesis method has many additional advantages as well, including low cost, flexibility in sequence and length designs, adaptivity to conventional solid-phase polynucleotide synthesis, and low chip-to-chip variability.
In addition to the use in polynucleotide synthesis, array fabrication methods in the present invention may also be used in in situ synthesis of other molecules including biopolymers such as polypeptides, polysaccharides, etc. The fabricated arrays may also be used as platforms for simultaneously carrying out large numbers of reactions, in particular, chemical and biological reactions. The present invention also features a method for reducing undesirable background signals in array-based applications.
The present invention features a method for fabricating a solid support comprising the steps of: (a) reacting a support surface with a first reagent to form a hydrophilic surface; (b) coating the support surface with a photoresist substance; (c) exposing selected regions of the support surface to light; (d) developing the support surface to form a patterned exposed surface and photoresist coated surface; (e) reacting the exposed surface with a second reagent to form hydrophobic sites; and (f) removing the photoresist coat from the photoresist coated surface.
The present invention also features a method for fabricating solid supports comprising the steps of: (a) reacting a support surface with a first reagent to form a hydrophobic surface; (b) coating the support surface with a photoresist substance; (c) exposing selected regions of the support surface to light; (d) developing the support surface to form a patterned exposed surface and photoresist coated surface; (e) removing the photoresist coat from the photoresist coated surface; and (f) reacting the exposed surface with a second reagent to form hydrophilic sites.
Any suitable solid supports may be used in the present invention. These materials include glass, silicon, wafer, polystyrene, polyethylene, polypropylene, polytetrafluorethylene, among others. Typically, the density of derivatized, hydrophilic or in situ synthesis sites on an array is between about 1-10,000 per cm2, preferably below about 5,000, 1,000, 400, 200, 100, or 60 per cm2. The area of each site may be about 0.1xc3x9710xe2x88x925 to 0.1 cm2, preferably less than about 0.05, 0.01, or 0.005 cm2. Typically, the total number of these sites on an array is between about 10-500,000, preferably, between about 10-100,000, 10-50,000, 10-10,000, 10-5000, 10-1000, 10-500, or 10-100. Synthesis reagents may be delivered using an ink-jet printing apparatus, such as a piezoelectric pump, a capillary tube, etc.
Fabricated solid supports may be further functionalized to provide covalent or noncovalent attachment to chemical or biological entities. In particular, fabricated/functionalized solid supports may be used in in situ synthesis of compounds, such as polynucleotides, polypeptides, or polysaccharides. Presynthesized compounds may also be deposited on these solid supports either covalently or non-covalently. Fabricated/functionalized solid supports may be employed as platforms for simultaneously carrying out large numbers of reactions. Any suitable unimolecular or non-unimolecular reaction (two or more reactants) may be applicable to the instant invention. For example, these reactions may involve cells, viruses, nucleic acids, proteins, peptides, carbohydrates, lipids, small molecules, etc. These reactions may be useful in the study, diagnosis, and treatment of genetic disorders, in genetic testing, in agriculture, in industrial use, etc.
The instant methods are particularly suited for in situ polynucleotide synthesis. It is possible to in situ synthesize polynucleotides of more than 15 nucleotides long with greater percentage of polynucleotides with the desired length at an in situ synthesis site. The average step yields are near or above about 98%. The present invention features solid supports comprising in situ synthesized polynucleotides wherein above about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of polynucleotides at an in situ synthesis site (e.g., hydrophilic sites) are longer than about 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides long.
The present method also features a method for reducing background signals from an array comprising the sequential steps of: (a) derivatizing a solid support to form hydrophilic or hydrophobic sites; (b) coating said hydrophilic or hydrophobic sites with a photoresist substance; and (c) removing said photoresist substance. By removing the residual amount of photoresist, the present method also allows the reduction of background signals from the array surface in hybridization assays or other array applications.